The elemental content of different compartments of Trypanosoma cruzi epimastigotes has been measured by quick freezing, ultracryomicrotomy, x-ray microanalysis and compositional mapping in the scanning transmission electron microscope. Vacuoles identified by high electron-density contained large amounts of phosphorus, and the divalent cations, magnesium, calcium and zinc. No other compartment had appreciable calcium or zinc content. Iron was detected only in vacuoles distinct from the electron-dense vacuoles and other organelles. To determine the distribution of calcium-rich and iron-rich vacuoles within the trypanosomes, compositional maps were collected using electron energy loss spectrum-imaging. In this technique, spectra were analyzed at each pixel to obtain distributions of calcium and iron. Incubation of cells in culture medium with ionomycin plus nigericin led to factor of 2-3 increase in potassium in the electron-dense vacuoles and in the iron-rich vacuoles. This finding indicated the acidic nature of the vacuoles and demonstrated that the electron-dense vacuoles correspond to the acidocalcisome, i.e., an acidic compartment rich in Ca2+, that has been characterized biochemically but has not been identified morphologically. The x-ray analysis and elemental mapping provide evidence that acidocalcisomes are organelles different from lysosomes or other organelles previously described in these parasites which are the agent causing Chagas' disease. This is a continuation of Intramural Research Project Z01-RR-10479-02 BEI.